We have been interested in defining the major mechanisms of simultaneous resistance of cancer cells to multiple chemotherapeutic agents. One major mechanism is expression of an energy-dependent efflux pump, termed P-glycoprotein (P-gp), or the multidrug transporter, encoded in humans by the MDR1 gene. The sequence of the MDR1 cDNA led to a model of the transporter as a pump with 12 transmembrane domains and 2 ATP sites; determination of the domains of P-gp responsible for substrate binding and coupling of ATPase activity to substrate transport are the major goals of our work. Model systems based on stable expression or transient expression of mutated P-gps by a vaccinia virus expression system have been developed to assay functional effects of these mutations on drug binding, drug-dependent ATPase, drug resistance and drug transport. Substitution of all known phosphorylation sites in P-gp with either Ala or Asp does not affect ability of P-gp to confer multidrug resistance. The creation of bicistronic retroviral expression vectors able to confer multidrug resistance has enabled the development of vectors for treatment of other genetic diseases such as Fabry disease, Gaucher disease, chronic granulomatous disease, X-linked severe combined immunodeficiency and adenosine deaminase deficiency. In these vectors, P-gp serves as a dominant selectable marker. These vectors may be delivered to bone marrow stem cells grown ex vivo or complexed to liposomes in vivo. We have also begun to explore the mechanism of multidrug resistance resulting from selection in cisplatin of hepatoma cells and KB adenocarcinoma cells. Cisplatin-resistant hepatoma and KB cells are cross-resistant to methotrexate, arsenite and antimonite and accumulate reduced amounts of these three toxic agents.